| 1. |
- Holm, Cecilia Koskinen, et al.
(författare)
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Lack of SIRP alpha phosphorylation and concomitantly reduced SHP-2-PI3K-Akt2 signaling decrease osteoblast differentiation
- 2016
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 478:1, s. 268-273
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Tidskriftsartikel (refereegranskat)abstract
- Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on intricate cellular signaling pathways, including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRP alpha) have both been suggested to regulate bone cell differentiation. Here we investigated osteoblastic differentiation of bone marrow stromal cells from SIRP alpha mutant mice lacking the cytoplasmic signaling domain of SIRPa. An impaired osteoblastogenesis in SIRP alpha-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. This reduced osteoblastic differentiation potential in SIRPa-mutant stromal cells was associated with a significantly reduced expression of Runx2, osterix, osteocalcin, and alkaline phosphatase mRNA, as well as a reduced phosphorylation of SHP-2 and Akt2, as compared with that in wild-type stromal cells. Addition of a PI3K-inhibitor to wild-type stromal cells could mimic the impaired osteoblastogenesis seen in SIRP alpha-mutant cells. In conclusion, our data suggest that SIRPa signaling through SHP-2-PI3K-Akt2 strongly influences osteoblast differentiation from bone marrow stromal cells.
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| 2. |
- Ikeda, Hiroshi, et al.
(författare)
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Mutational analysis of the mechanism of negative regulation by SRC homology 2 domain-containing protein tyrosine phosphatase substrate-1 of phagocytosis in macrophages.
- 2006
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Ingår i: Journal of Immunology. - 0022-1767. ; 177:5, s. 3123-32
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Tidskriftsartikel (refereegranskat)abstract
- Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is a transmembrane protein predominantly expressed in macrophages. The binding of CD47 on RBCs to SHPS-1 on macrophages is implicated in inhibition of phagocytosis of the former cells by the latter. We have now shown that forced expression in mouse RAW264.7 macrophages of a mutant version (SHPS-1-4F) of mouse SHPS-1, in which four tyrosine phosphorylation sites are replaced by phenylalanine, markedly promoted Fc gammaR-mediated phagocytosis of mouse RBCs or SRBCs. Forced expression of another mutant form (SHPS-1-deltaCyto) of mouse SHPS-1, which lacks most of the cytoplasmic region, did not promote such phagocytosis. Similarly, forced expression of a rat version of SHPS-1-4F, but not that of rat wild-type SHPS-1 or SHPS-1-deltaCyto, in RAW264.7 cells enhanced Fc gammaR-mediated phagocytosis of RBCs. Tyrosine phosphorylation of endogenous SHPS-1 as well as its association with Src homology 2 domain-containing protein tyrosine phosphatase-1 were not markedly inhibited by expression of SHPS-1-4F. Furthermore, the attachment of IgG-opsonized RBCs to RAW264.7 cells was markedly increased by expression of SHPS-1-4F, and this effect did not appear to be mediated by the interaction between CD47 and SHPS-1. These data suggest that inhibition by SHPS-1 of phagocytosis in macrophages is mediated, at least in part, in a manner independent of the transinteraction between CD47 and SHPS-1. In addition, the cytoplasmic region as well as tyrosine phosphorylation sites in this region of SHPS-1 appear indispensable for this inhibitory action of SHPS-1. Moreover, SHPS-1 may regulate the attachment of RBCs to macrophages by an as yet unidentified mechanism.
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| 3. |
- Ishikawa-Sekigami, Tomomi, et al.
(författare)
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Enhanced phagocytosis of CD47-deficient red blood cells by splenic macrophages requires SHPS-1.
- 2006
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 343:4, s. 1197-200
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Tidskriftsartikel (refereegranskat)abstract
- The interaction of CD47 on red blood cells (RBCs) with SHPS-1 on macrophages is implicated to prevent the phagocytosis of the former cells by the latter cells. Indeed, the rate of clearance of transfused CD47-deficient (CD47(-/-)) RBCs from the bloodstream of wild-type mice was markedly increased compared with wild-type RBCs. Conversely, the rate of clearance of transfused wild-type RBCs was markedly increased in mice that expressed a mutant form of SHPS-1 lacking most of the cytoplasmic region of the protein. However, we here found that the clearance of CD47(-/-) RBCs in SHPS-1 mutant mice was minimal. In addition, the phagocytosis of CD47(-/-) RBCs by splenic macrophages from SHPS-1 mutant mice was markedly reduced compared with wild-type macrophages. These results thus suggest an additional role for CD47 on RBCs in the negative regulation of phagocytosis by macrophages and in determination of the life span of circulating RBCs.
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| 4. |
- Kolan, Shrikant, 1983-, et al.
(författare)
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Lack of non-hematopoietic SIRPα signaling disturbs the splenic marginal zone architecture resulting in accumulation and displacement of marginal zone B cells
- 2015
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 460:3, s. 645-650
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Tidskriftsartikel (refereegranskat)abstract
- Signal regulatory protein α (SIRPα) is an immunoglobulin super family protein predominantly expressed by myeloid but not lymphoid cells, and its role in lymphocyte homeostasis and function is still to be revealed. We demonstrate that mice bearing a mutant SIRPα lacking the cytoplasmic signaling domain (SIRPα MT) had an increased amount of splenic marginal zone (MZ) B cells compared to wild-type controls. Immunohistochemical analysis revealed an increased localization of MZB cells into B cell follicular areas of the white pulp in SIRPα MT spleens. However, we found no signs of an increased MZB cell activation level in MT mice. The immune response to T-independent antigens in vivo was slightly increased in SIRPα MT mice while sorted MZB from these mice responded normally to LPS in vitro. Bone marrow reconstitution experiments demonstrated that the MZB cell phenotype of SIRPα MT mice was due to lack of SIRPα signaling in non-hematopoietic cells. In contrast, MZ retention of MZ macrophages required hematopoietic SIRPα, while normal distribution of metallophilic macrophages required non-hematopoietic SIRPα signaling. In summary, these data identified SIRPα signaling in non-hematopoietic cells to play an important role in regulating the numbers and positioning MZB cell in the spleen.
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| 5. |
- Kolan, Shrikant, 1983-, et al.
(författare)
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Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen
- 2015
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Ingår i: PLoS One. - : plos one. - 1932-6203. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlofollicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.
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| 6. |
- Koskinen, Cecilia, et al.
(författare)
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Lack of CD47 impairs bone cell differentiation and results in an osteopenic phenotype in vivo due to impaired signal regulatory protein α (SIRPα) signaling
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:41, s. 29333-29344
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Tidskriftsartikel (refereegranskat)abstract
- Here, we investigated whether the cell surface glycoprotein CD47 was required for normal formation of osteoblasts and osteoclasts and to maintain normal bone formation activity in vitro and in vivo. In parathyroid hormone or 1α,25(OH)2-vitamin D3 (D3)-stimulated bone marrow cultures (BMC) from CD47(-/-) mice, we found a strongly reduced formation of multinuclear tartrate-resistant acid phosphatase (TRAP)(+) osteoclasts, associated with reduced expression of osteoclastogenic genes (nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp). The production of M-CSF and RANKL (receptor activator of nuclear factor κβ ligand) was reduced in CD47(-/-) BMC, as compared with CD47(+/+) BMC. The stromal cell phenotype in CD47(-/-) BMC involved a blunted expression of the osteoblast-associated genes osterix, Alp/Akp1, and α-1-collagen, and reduced mineral deposition, as compared with that in CD47(+/+) BMC. CD47 is a ligand for SIRPα (signal regulatory protein α), which showed strongly reduced tyrosine phosphorylation in CD47(-/-) bone marrow stromal cells. In addition, stromal cells lacking the signaling SIRPα cytoplasmic domain also had a defect in osteogenic differentiation, and both CD47(-/-) and non-signaling SIRPα mutant stromal cells showed a markedly reduced ability to support osteoclastogenesis in wild-type bone marrow macrophages, demonstrating that CD47-induced SIRPα signaling is critical for stromal cell support of osteoclast formation. In vivo, femoral bones of 18- or 28-week-old CD47(-/-) mice showed significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. In conclusion, lack of CD47 strongly impairs SIRPα-dependent osteoblast differentiation, deteriorate bone formation, and cause reduced formation of osteoclasts.
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| 7. |
- Koskinen, Cecilia, 1979-, et al.
(författare)
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Reduced SIRPα phosphorylation and concommitant SHP-2–PI3K–Akt2 signaling decrease osteoblast differentiation
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on an intricate cellular signaling including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) are suggested to regulate bone cell differentiation. In the present study, we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. Moreover, we compared downstream signaling events of SIRPα in wild-type and CD47-deficient mouse bone marrow stromal cells. SIRPα-mutant stromal cells showed significantly less expression of Runx2, Sp7 (osterix), Bglap (osteocalcin), and Akp1 (alkaline phosphatase) mRNA compared to stromal cells from wild-type mice. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. Western blot analyses showed that CD47 expression was required for Src homology-2 domain containing protein tyrosine phosphatase- 2 (SHP-2) to associate with SIRPa. As a result, SHP-2 and Akt2 in stromal cells from CD47 deficient mice were less phosphorylated, as compared to that in wild-type stromal cells. In conclusion, we here show that CD47-dependent SIRPα signaling through SHP-2–PI3K–Akt2 strongly influences osteogenic differentiation of bone marrow stromal cells.
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| 8. |
- Marschinke, Franziska, et al.
(författare)
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The absence of CD47 promotes nerve fiber growth from cultured ventral mesencephalic dopamine neurons
- 2012
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Ingår i: PLOS ONE. - : Public library of science. - 1932-6203. ; 7:9, s. e45218-
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Tidskriftsartikel (refereegranskat)abstract
- In ventral mesencephalic organotypic tissue cultures, two timely separated sequences of nerve fiber growth have been observed. The first appearing nerve fiber pattern is a long-distance outgrowth that occurs before astrocytes start to proliferate and migrate to form an astrocytic monolayer that finally surrounds the tissue slice. These long-distance growing nerve fibers are retracted as the astrocytes migrate, and are followed by a secondary outgrowth. The secondary outgrowth is persistent in time but reaches short distances, comparable with outgrowth seen from a dopaminergic graft implanted to the brain. The present study was focused on the interaction between the astrocytes and the long-distance growing non-glial associated nerve fibers. Cross talk between astroglia and neurite formation might occur through the integrin-associated protein CD47. CD47 serves as a ligand for signal regulatory protein (SIRP) alpha and as a receptor for the extracellular matrix protein thrombospondin-1 (TSP-1). Embryonic day 14 ventral mesencephalic tissue from CD47(+/+) and CD47(-/-) mice was used to investigate astrocytic migration and the tyrosine hydroxylase (TH) -positive outgrowth that occurred remote from the astrocytes. TH-immunohistochemistry demonstrated that the non-glial-associated nerve fiber outgrowth in CD47(-/-) cultures reached significantly longer distances and higher density compared to nerve fibers formed in CD47(+/+) cultures at 14 days in vitro. These nerve fibers often had a dotted appearance in CD47(+/+) cultures. No difference in the astrocytic migration was observed. Further investigations revealed that the presence of CD47 in control culture did neither hamper non-glial-associated growth through SIRP alpha nor through TSP-1 since similar outgrowth was found in SIRP alpha mutant cultures and in CD47(+/+) cultures treated with blocking antibodies against the TSP-1, respectively, as in the control cultures. In conclusion, long-distance growing nerve fiber formation is promoted by the absence of CD47, even though the presence of astrocytes is not inhibited.
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| 9. |
- Maruyama, Toshi, et al.
(författare)
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Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain
- 2012
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Ingår i: Journal of Neurochemistry. - : John Wiley & Sons. - 0022-3042 .- 1471-4159. ; 121:6, s. 891-902
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Tidskriftsartikel (refereegranskat)abstract
- Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37°C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature (T(rec) ) of mice was reduced to 27° to 30°C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test.
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| 10. |
- Meinderts, Sanne M., et al.
(författare)
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Human and murine splenic neutrophils are potent phagocytes of IgG-opsonized red blood cells
- 2017
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 1:14, s. 875-886
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Tidskriftsartikel (refereegranskat)abstract
- Red blood cell (RBC) clearance is known to occur primarily in the spleen, and is presumed to be executed by red pulp macrophages. Erythrophagocytosis in the spleen takes place as part of the homeostatic turnover of RBCs to remove old RBCs. It can be strongly promoted by immunoglobulin G (IgG) opsonization of RBCs, a condition that can occur as a consequence of autoantibody or alloantibody formation. The purpose of our study was to investigate which phagocytes are involved in IgG-mediated RBC clearance in the human spleen. We developed a highly specific in vitro assay to monitor RBC phagocytosis in total human splenocytes. Surprisingly, we have found that whereas homeostatic clearance of RBCs is primarily a task for splenic macrophages, neutrophils and, to a lesser extent, also monocytes can be a major factor in clearance of IgG-opsonized RBCs. Erythrophagocytosis by neutrophils is strongly dependent on the degree of opsonization of the RBCs. Additionally, the process is enhanced after blocking the "do not eat me" signal CD47 on the opsonized RBCs, which binds signal regulatory protein a, a myeloid inhibitory receptor that restricts phagocytosis. Moreover, RBCs isolated from autoimmune hemolytic anemia patients, opsonized by auto-IgGs, were shown to be readily phagocytosed by neutrophils. Finally, priming of neutrophils by inflammatory mediators such as tumor necrosis factor a and lipopolysaccharide further increases the magnitude of erythrophagocytosis. Collectively, our data suggest that neutrophils contribute significantly to the phagocytosis of antibody-opsonized RBCs, especially under inflammatory conditions. This indicates a hereto unanticipated contribution of neutrophils in RBC phagocytosis, especially under pathological conditions such as alloimmunization or autoimmunization.
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